Studies on the availability of intramitochondrial carbamoylphosphate for utilization in extramitochondrial reactions in rat liver

The following observations with isolated mitochondria prepared from rat liver demonstrate that Carbamoylphosphate can readily traverse the mitochondrial membrane: (a) Citrulline synthesis occurs within isolated intact mitochondria at the expense of exogenously added ornithine and [¹⁴C]carbamoylphosphate, providing evidence that the initochondrial membrane does not exclude extramitochondrial car bamoylphosphate from penetrating the intramitochondrial matrix, (b) The [¹⁴C]carbamoylphosphate synthesized within isolated intact mitochondria from NaH¹⁴CO₃ by the action of the N-acetyl-l-glutamate-activated carbamoylphosphate synthetase (CPS-I) is equally available for consumption in intramitochondrial and extramitochondrial reactions, as judged by the coupled activity of CPS-I with either intramitochondrial ornithine carbamoyltransferase or extramitochondrial aspartate carbamoyltransferase. The possibility that the coupled action of CPS-I with intramitochondrial ornithine carbamoyltransferase might prevent the export of carbamoylphosphate into the extramitochondrial medium was also examined. The addition of ornithine to the reaction mixture, at concentrations which are optimal for citrulline production, did not reduce carbamoylphosphate export below$\text{1}\text{3}$ of the total amount of carbamoylphosphate synthesized. These results indicate that the carbamoylphosphate generated intramitochondrially is not compartment ally excluded from participating in cytoplasmic reactions, and raise the possibility that the intramitochondrial carbamoylphosphate synthetase, CPS-I, may be a significant source of the carbamoylphosphate incorporated into hepatic pyrimidines by the cytoplasmic enzymes of the orotate pathway.     

Regulation of aspartate carbamoyltransferase of Escherichia coli by the interrelationship of magnesium and nucleotides.

Purified aspartate carbamoyltransferase from Escherichia coli K12 (carbamoylphosphate: L-aspartate carbamyltransferase, EC 2.1.3.2) shows greater activity with nucleotide effectors as the magnesium nucleotide complex than with similar amounts of the sodium nucleotide. Regulation of aspartate carbamoyltransferase activity in vivo may occur by changes in the total concentration of regulatory nucleotides or, under conditions of magnesium-limited growth, by variation of the saturation of the nucleotides with magnesium.     

Characterization of the aspartate carbamoyltransferase fragment generated by protease action on the pyrimidine-3 gene product of Neurospora crassa.

The molecular weight of the fragment of aspartate carbamoyltransferase (carbamoylphosphate: L-aspartate carbamoyltransferase, EC 2.1.3.2) of Neurospora crassa following proteolysis was found to be 1.0-10(5) (aspartate carbamoyltransferase-L). It differs from the native form of the enzyme (aspartate carbamoyltransferase-N, 6.5-10(5)) in several respects. It has a lower V, has a much greater affinity (approx. 3-fold) for L-aspartate, and is strongly activated by glycine. Both forms of aspartate carbamyoltransferase have a pH optimum of approx. 9.5, and they exhibit similar affinities for carbamoyl phosphate.     

pryB mutations as suppressors of arginine auxotrophy in Salmonella typhimurium.

Salmonella typhimurium strains which produce high constitutive levels of aspartate transcarbamoylase due to the pyrH700 mutation were found to grow more slowly in minimal medium than pyrH+ controls. The addition of arginine or citrulline but not ornithine restored normal growth rates. This requirement for arginine was completely suppressed by pyrB mutations and partially suppressed by pyrC and pyrD mutations. No suppression was observed with mutants at the pyrF locus. Introduction of leaky mutation argI2002 resulted in a more extreme arginine requirement and accentuated suppression by pyrB mutations. Suppression by the pyrC and pyrD mutations was reduced as a result of the incorporation of the leaky argI2002 allele. These results indicate that in pyrH700 strains carbamoyl phosphate is preferentially directed toward the formation of intermediates in the pyrimidine biosynthetic pathway. Arginine auxotrophy results from the reduced availability of carbamoyl phosphate for the biosynthesis of arginine. Suppression of this arginine dependence for growth is used as a convenient positive selection technique for pyrB mutations.     

Isolation and heteroduplex mapping of a lambda transducing bacteriophage carrying the structural genes for carbamoylphosphate synthase: regulation of enzyme synthesis in Escherichia coli K-12 lysogens.

A N-lambda bacteriophage transducing the structural genes for Escherichia coli K-12 carbamoylphosphate synthase (glutamine) (CPSase; EC 2.7.2.9) has been isolated and analyzed both genetically and physically. The whole int-N region is substituted for a short chromosomal segment corresponding almost exactly to the car locus. The study of CPSase, ornithine carbamoyltransferase, and aspartate carbamoyltransferase regulation in carriers of lambdadcar confirms the previously reported participation of the argR gene product in the control of CPSase synthesis and points to the existence of a regulatory molecule involved in the control of both CPSase and aspartate carbamoyltransferase synthesis. The general usefulness of using N- lambda transducing bacteriophages for the recovery of large amounts of gene products is discussed.     

Genetic characterization of the folding domains of the catalytic chains in aspartate transcarbamoylase

In Salmonella typhimurium strains which produce high constitutive levels of aspartate transcarbamoylase due to the pyrH700 mutation, the bulk of the carbamoyl phosphate of the cell is consumed for the biosynthesis of pyrimidines. As a consequence, there is little substrate available for arginine synthesis and the cell growth is impeded. Suppression of arginine auxotrophy by mutations which block aspartate transcarbamoylase activity provides a positive selection technique for mutant strains defective in this enzyme activity. A genetic analysis was performed on 29 mutant strains harboring defects in the structural gene pyrB, encoding the catalytic chains of aspartate transcarbamoylase of Escherichia coli. Extracts from 15 strains contained intact, inactive enzyme-like molecules of the same size as the purified wild type enzyme. These same extracts contained a predominant polypeptide chain which migrated electrophoretically at the same rate as catalytic chains from wild type enzyme. In addition to these 15 different missense mutants, 14 others (presumably chain-terminating mutants) were isolated; no polypeptides corresponding to full length catalytic chains were detected in these strains. Based on their reversion and suppression properties, seven were designated as frameshift and two as amber nonsense. A fine structure recombination map of the pyrB locus was constructed from a series of three-factor transductional crosses. Mutational sites were correlated with regions in the polypeptide sequence by relating their map positions to that of mutation pyrB231 which results in an amino acid replacement at position 128. Moreover, since recent crystallographic studies indicate that residue 128 is located near the junction between the NH2- and COOH-terminal folding domains, the mutational sites can be placed within either of these two regions of tertiary structure. Interallelic complementation experiments showed four units of complementation. Those defining the alpha and beta units were missense mutants with their mutational sites in the NH2- and COOH-terminal domains, respectively. The mutants determining the delta and gamma units involved premature polypeptide chain termination and their mutational sites were correlated with distal regions of the two respective domains. Several mutants of the chain-terminating type failed to complement members of more than one unit. Possible effects of the various mutations and their implications for mechanisms of complementation and enzyme activity are presented.     

The catalytic mechanism of Escherichia coli aspartate carbamoyltransferase: a molecular modelling study

Based on molecular modelling study, we propose that the reaction between L-aspartate an carbamoylphosphate, catalyzed by E. coli aspartate carbamoyltransferase, may proceed via a tetrahedral intermediate and that the breakdown of the intermediate is facilitated by an intramolecular proton transfer between the amino group of L-aspartate and a terminal phosphate oxygen of carbamoylphosphate.     

Evidence of carbamoylphosphate induced conformational changes upon binding to human ornithine carbamoyltransferase.

Human liver ornithine carbamoyltransferase undergoes absorbance changes in the UV region upon formation of the carbamoylphosphate-norvaline-enzyme ternary complex. The UV changes are similar in the presence of carbamoylphosphate alone, whilst they are lower in the presence of ornithine or norvaline alone. The extent of the UV changes correlates with the enzyme susceptibility to proteolytic degradation. The free native enzyme is completely and rapidly hydrolyzed by trypsin, whilst it is partially protected upon carbamoylphosphate binding. The extent of protection increases for the carbamoylphosphate-norvaline-enzyme ternary complex. These results strongly suggest that the binding of the first substrate, i.e. carbamoylphosphate, to human ornithine carbamoyltransferase induces a large protein isomerization, which regards the polar domain plus a part of equatorial domain of each subunit.     

Cyanate specifically inhibits arginine biosynthesis in Escherichia coli K12: a case of by-product inhibition?

Growth of Escherichia coli K12 cultivated in minimal medium was strongly inhibited by 2 mM-cyanate. This inhibition could be specifically reversed by arginine. Citrulline (but not ornithine, N-alpha-acetylornithine or N-acetylglutamate) could also restore a normal growth rate. Since growth inhibition by cyanate was followed by an accumulation of ornithine within the cell it was concluded that cyanate specifically inhibits the formation of citrulline from ornithine. The effect of cyanate on the growth of defined strains was consistent with a specific inhibition of carbamoylphosphate synthase. A kinetic study of carbamoylphosphate synthase and ornithine carbamoyltransferase in vitro supported this conclusion. Since carbamoylphosphate is probably the only source of endogenous cyanate it is postulated that carbamoylphosphate synthase activity can be regulated by cyanate resulting from the dissociation of carbamoylphosphate in metabolic circumstances leading to its overproduction.     

Investigation of binding sites in the complex pyrimidine-specific carbamoyl-phosphate synthetase/aspartate carbamoyltransferase enzyme of Neurospora crassa

The pyr-3 gene of Neurospora crassa codes for the bifunctional enzyme pyrimidine-specific carbamoyl-phosphate synthetase/aspartate carbamoyltransferase (carbon dioxide: ammonia ligase (ADP-forming, carbamate-phosphorylating)/carbamoylphosphate: L-aspartate carbamoyltransferase), EC 6.3.4.16/EC 2.1.3.2). We describe the investigation of substrate- and product-binding sites of the enzyme by affinity chromatography, using the ligands aspartate, glutamate, and adenosine 5'-diphosphate, and investigate the channelling of carbamoyl phosphate, the product of the first function and substrate of the second, through the pathway. For this latter aspect of the investigation, two new enzyme assays were devised and described. The results of the competition studies on carbamoyl phosphate-binding are consistent with the existence of two different binding sites within the enzyme for this metabolic intermediate, one for it as the product of the first step and the other for it as the substrate of the second.     

Methionine-321 in the C-terminal α-helix of catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa is important for positive homotropic cooperativity

Abstract Pseudomonas aeruginosa has a pair of distinct ornithine carbamoyltransferases. The anabolic ornithine carbamoyltransferase encoded by the argF gene catalyzes the formation of citrulline from ornithine and carbamoylphosphate. The catabolic ornithine carbamoyltransferase encoded by the arcB gene promotes the reverse reaction in vivo; although this enzyme can be assayed in vitro for citrulline synthesis, its unidirectionality in vivo is determined by its high concentration at half maximum velocity for carbamoylphosphate ([S]_0.5) and high cooperativity toward this substrate. We have mutant forms of catabolic ornithine carbamoyltransferase catalyzing the anabolic reaction in vivo. The corresponding arcB mutant alleles on a multicopy plasmid specifically suppressed an argF mutation of P. aeruginosa . Two new mutant enzymes were obtained. When methionine 321 was replaced by isoleucine, the mutant enzyme showed loss of homotropic cooperativity at physiological carbamoylphosphate concentrations. Substitution of glutamate 105 by lysine resulted in a partial loss of the sigmoidal response to increasing carbamoylphosphate concentrations. However, both mutant enzymes were still sensitive to the allosteric activator AMP and to the inhibitor spermidine. These results indicate that at least two residues of catabolic ornithine carbamoyltransferase are critically involved in positive carbamoylphisphate cooperativity: glutamate 105 (previously known to be important) and methionine 321. Mutational changes in either amino acid will affect the geometry of helix H2, which contains several residues required for carbamoylphosphate binding.     

Partial characterization of ornithine carbamoyltransferase in three microalgae : anabolic role only

Although the existence of isozymes of ornithine carbamoyltransferase (carbamoylphosphate:l-ornithine carbamoyltransferase, EC 2.1.3.3) in higher plants has been reported, and the possibility exists that one or more of these operates catabolically to produce ornithine and carbamoylphosphate from citrulline and inorganic phosphate, no proof has been forthcoming. In view of the fact that many unicellular algae degrade arginine via arginine deiminase to citrulline and ammonium, and that the pathway of utilization of citrulline is unknown, we decided to investigate the possibility of the presence of a catabolic form of ornithine carbamoyltransferase in three microalgae known to have arginine deiminase activity. These were Chlorella autotrophica, Chlorella saccharophila, and Dunaliella tertiolecta. Our results show that the properties of OCT from these three algae are similar to OCTs from many higher plants with respect to general kinetics (K_m values for ornithine and carbamoylphosphate), substrate inhibition by ornithine at high pHs, apparent sequential ordered kinetic mechanisms and paucity of apparent regulatory properties. Our data indicate an exclusively anabolic role of ornithine carbamoyltransferase in these algae.     

Increase in arginine and citrulline production by 6-azauracil-resistant mutants of Bacillus subtilis.

In the arginine producer AHr-5, an L-arginine hydroxamate-resistant mutant of Bacillus subtilis, accumulation of N8-acetyl-L-ornithine increased as the level of L-arginine accumulation increased in the medium containing L-glutamic acid. Ornithine carbamoyltransferase of this strain was genetically derepressed. These results suggested that carbamoylphosphate might be deficient in vivo. With the intention to increase endogenous carbamoylphosphate, pyrimidine analogs inhibiting growth were selected and the mutants resistant to these compounds were derived from the AHr-5 mutant. Of the resistant mutants derived, the 6-azauracil-resistant mutant AAr-9 produced 28 mg of L-arginine per ml, which corresponded to more than twofold the amount produced by the parent strain. Derivation of an arginine-requiring mutant from the double-resistant mutant AAr-9 provides a new advantageous method for the production of L-citrulline. The increase in arginine and citrulline production is discussed.     

Increase in arginine and citrulline production by 6-azauracil-resistant mutants of Bacillus subtilis.

In the arginine producer AHr-5, an L-arginine hydroxamate-resistant mutant of Bacillus subtilis, accumulation of N8-acetyl-L-ornithine increased as the level of L-arginine accumulation increased in the medium containing L-glutamic acid. Ornithine carbamoyltransferase of this strain was genetically derepressed. These results suggested that carbamoylphosphate might be deficient in vivo. With the intention to increase endogenous carbamoylphosphate, pyrimidine analogs inhibiting growth were selected and the mutants resistant to these compounds were derived from the AHr-5 mutant. Of the resistant mutants derived, the 6-azauracil-resistant mutant AAr-9 produced 28 mg of L-arginine per ml, which corresponded to more than twofold the amount produced by the parent strain. Derivation of an arginine-requiring mutant from the double-resistant mutant AAr-9 provides a new advantageous method for the production of L-citrulline. The increase in arginine and citrulline production is discussed.     

Repression and derepression of the enzymes of the pyrimidine biosynthetic pathway in Salmonella typhimurium

Activities of five enzymes of the pyrimidine biosynthetic pathway and one enzyme involved in arginine synthesis were measured during batch culture of Salmonella typhimurium. Aspartate carbamoyltransferase, dihydroorotase, and the arginine pathway enzyme, ornithine carbamoyltransferase, remained constant during the growth cycle but showed a sharp decrease in activity after entering the stationary phase. Dihydroorotate dehydrogenase, orotate phosphoribosyltransferase and orotidine-5'-monophosphate (OMP) decarboxylase showed peaks of activity corresponding to the mid-point of the exponential phase of growth while remaining comparatively stable in the stationary phase. Derepression studies carried out by starving individual pyrimidine (Pyr-) deletion mutants for uracil showed that the extent of derepression obtained for aspartate carbamoyltransferase, dihydroorotase and dihydroorotate dehydrogenase depended on the location of the pyr gene mutation. Orotate phosphoribosyltransferase and OMP decarboxylase derepression levels were independent of the location of the pyr mutation. Aspartate carbamoyltransferase showed the greatest degree of derepression of the six enzymes studied, with pyrA strains (blocked in the first step of the pathway) showing about twice as much derepression as pyrF strains (blocked in the sixth step of the pathway). A study of the kinetics of repression on derepressed levels of the pyrimidine enzymes produced data that were compatible with dilution of specific activity by cell division when repressive amounts of uracil were added to the derepression medium.     

Organization of a multifunctional protein in pyrimidine biosynthesis. A domain hypersensitive to proteolysis.

When the multifunctional protein that catalyses the first three steps of pyrimidine biosynthesis in hamster cells is treated with staphylococcal V8 proteinase, a single cleavage takes place. The activities of carbamoyl-phosphate synthetase (EC 6.3.5.5), aspartate carbamoyltransferase (EC 2.1.3.2) and dihydro-orotase (EC 3.5.2.3) and the allosteric inhibition by UTP are unaffected. One fragment, of Mr 182000, has the first and third enzyme activities, whereas the other fragment, of Mr 42000, has aspartate carbamoyltransferase activity and an aggregation site. A similar small fragment is observed in protein digested with low concentrations of trypsin. A similar large fragment is seen after digestion with trypsin and as the predominating form of this protein in certain mutants defective in pyrimidine biosynthesis. These results indicate that a region located adjacent to the aspartate carbamoyltransferase domain is hypersensitive to proteinase action in vitro and may also be sensitive to proteolysis in vivo.     

Localization of carbamoylphosphate synthetase and aspartate carbamoyltransferase in chloroplasts

The localization of carbamoylphosphate synthetase (CPSase) and aspartate carbamoyltransferase (ACTase), the first two enzymes of the pyrimidine biosynthetic pathway, in chloroplasts was investigated. In dark-grown radish (Raphanus sativus) seedlings, light induced a prominent increase in CPSase activity, but had little effect on ACTase activity. Both enzymes were found in chloroplasts isolated from radish cotyledons and leaves of spinach (Spinacia oleracea), soybean (Glycine max), and corn (Zea mays). The higher activity of ACTase relative to CPSase is discussed in relation to the instability of carbamoylphosphate, the product of the CPSase, and to the control of pyrimidine synthesis. Based on these results, the function of CPSase and ACTase in chloroplasts is discussed.     

Anabolic ornithine carbamoyltransferase of Escherichia coli and catabolic ornithine carbamoyltransferase of Pseudomonas putida. Steady-state kinetic analysis.

The anabolic and catabolic ornithine carbamoyltransferases of Pseudomonas putida display an undirectional catalytic specialization: in citrulline synthesis for the anabolic enzyme, in citrulline phosphorolysis for the catabolic one. The irreversibility of the anabolic enzyme in vitro has been previously explained by its kinetic properties, whereas the irreversibility of the catabolic transferase in vivo was shown to be due to its allosteric behaviour. In this work a steady-state kinetic analysis has been carried out on the catabolic ornithine carbamoyltransferase at pH 6.8 in the presence of the allosteric activator, phosphate. The kinetic mechanism of Escherichia coli ornithine carbamoyltransferase serving as a reference was also determined. For the E. coli enzyme in the reverse direction, the initial velocity patterns converging on the abscissa were obtained with either citrulline or arsenate as variable substrate. The inhibition by the product ornithine was linear competitive with respect to citrulline and linear non-competitive with respect to arsenate. In the forward direction phosphate and its analogs induce an inhibition by ornithine which is partial and competitive with respect to carbamoylphosphate. Together with the results of thermo-inactivation studies in the presence of each reactant, this observation suggests a random kinetic mechanism, but with most of the reaction flux following the path where carbamoylphosphate adds before ornithine, when substrates are present at Km levels. The allosteric catabolic ornithine carbamoyltransferase of Pseudomonas displays qualitatively the same pattern as the E. coli enzyme.     

Structure and properties of the putrescine carbamoyltransferase of Streptococcus faecalis.

Ornithine and putrescine carbamoyltransferases from Streptococcus faecalis ATCC11700 have been purified and their structural properties compared. The molecular weight of native ornithine carbamoyltransferase, measured by molecular sieving, is 250 000. It is composed of six apparently identical subunits with a molecular weight of 39 000, as determined by cross-linking with the bifunctional reagent glutaraldehyde followed by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. Using the same method, putrescine carbamoyltransferase is a trimer of 140 000 consisting of three identical subunits with a molecular weight of 40 000. Ornithine carbamoyltransferase displays a narrow specificity towards its substrate, ornithine. In contrast, putrescine carbamoyltransferase carbamoylates ornithine and several diamines (diaminopropane, diaminohexane, spermine, spermidine, cadaverine) in addition to its preferred substrate, putrescine, but with a considerable lower efficiency than for putrescine. The kinetic mechanism of putrescine carbamoyltransferase has been investigated. Initial velocity studies yield intersecting plots using either putrescine or ornithine as substrate, indicating a sequential mechanism. The patterns of protection of the enzyme by the reactants during heat inactivation as well as the results of product and dead-end inhibition studies provide evidence for a random addition of the substrates. The putrescine inhibition that is induced by phosphate does, however, suggest that a preferred pathway exists in which carbamoylphosphate is the leading substrate. The different kinetic constants have been established. The properties of putrescine carbamoyltransferase are compared to the known properties of other carbamoyltransferases. The evolutionary implications of this comparison are discussed.